Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Abstract

SM. Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress. Am J Physiol Lung Cell Mol Physiol 295: L756 -L766, 2008. First published August 29, 2008 doi:10.1152/ajplung.00146.2007.-Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 g/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO x), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P Ͻ 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P Ͻ 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P Ͻ 0.05). Conversely, NO x levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P Ͻ 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P Ͻ 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P Ͻ 0.05) and were greater in shunted lambs than controls at all ages (P Ͻ 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P Ͻ 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO. pulmonary circulation; oxidant stress; congenital heart disease; reactive oxygen species INFANTS AND CHILDREN with congenital cardiac defects that cause significantly increased pulmonary blood flow suffer morbidity due to early aberrations in pulmonary vascular function (17). In fact, this early pulmonary vascular dysfunction is often exacerbated in the immediate postoperative period, manifesting as increased vascular reactivity that may produce severe hypoxemia, acidosis, low cardiac output, and death if not treated immediately (7,11,31). A complete understanding of the mechanisms responsible for this pulmonary vascular dysfunction is lacking, but evidence suggests that aberrant nitric oxide (NO)-cGMP signaling and oxidative stress may participate (1,3,6,11,15,33,39,41).Basal NO production by the vascular endothelium is integral to the maintenance of the normal low resistance state of the pulmonary vasculature, and dynamic alterations in NO production modulate vascular relaxation and constriction in response to various stimuli. NO is produced in ...

Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow

“…Therefore we conclude that pulmonary capillary hydrostatic pressure in acute and chronic shunt lambs was elevated due to increased PBF. Additionally, because the lymph-to-plasma-protein ratio declined to a similar extent in the acute and chronic models, chronic exposure to increased PBF did not result in measurable losses in pulmonary capillary barrier function, despite previously described pulmonary vascular endothelial dysfunction (52).…”

Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

“…To detect superoxide generation in peripheral lung tissue, EPR measurements were performed using the spin trap, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine .HCl as already described (35,45). NOS-derived superoxide levels were determined by subtracting the superoxide values in the presence of the NOS inhibitor, ethylisothiourea (100 lM, ETU) from the superoxide values in the absence of ETU.…”

Mitochondrial Dysfunction and Congenital Heart Disease

“…Previous work has shown that eNOS-hsp90 interactions are progressively disrupted during the development of the endothelial dysfunction associated with increased PBF [75]. In addition to eNOS, hsp90 also chaperones other proteins involved in NO signaling.…”

“…Thus, the loss of ATP due to mitochondrial dysfunction attenuates hsp90 activity resulting in a "feed-forward" signaling cascade in which eNOS becomes ever more uncoupled leading to less NO-and more peroxynitrite-being produced driving pulmonary endothelial dysfunction [75] (Figure 3). In addition, the NO receptor, soluble Guanylyl Cyclase (sGC) is also found in a complex with hsp90 and Inhibition of hsp90 activity using geldanamycin stimulates it proteasomal degradation again via CHIP reducing the ability of the cell to produce cyclic guanosine monophosphate (cGMP) [77][78][79][80].…”

“…Congenital heart defects with increased PBF are associated with the development of increased pulmonary vascular reactivity and pulmonary hypertension [1][2][3][4]. In the post-natal period, the presence of large communications at the level of the ventricles (e.g.…”